Peptide/Amide Bond Strategies With OxymaPure®
Yield and racemization during the formation of Z-Phg-Pro-NH2 in DMF (solution synthesis).[a] (2)
| Entry | Coupling reagent/strategy | Yield (%) | D/L (%) |
| 1 | HOAt/DIC | 81.4 | 3.3 |
| 2 | HOBt/DIC | 81.9 | 9.3 |
| 3 | Oxyma/DIC | 89.9 [b] | 1.0 |
| 4 | Oxyma/DIC | 88.2 [c] | 1.1 |
| 5 | HOPO/DIC | 88.2 | 17.4 [d] |
| 6 | HOPO/DIC | 83.4 [c] | 6.1 |
[a] Couplings were conducted without preactivation, except for entries 4 and 6. The tR values of LL and DL were identified by co-injection with pure samples. [b] Unreacted Z-Phg-OH was detected. [c] A 2 min preactivation time was used. [d] Extra peak was found at 22.4 min (6.0%)
Yield and racemization during the formation in solution of Z-Phe-Val-Pro-NH2 in DMF in solution from Z-Phe-Val-OH and H-Pro-NH2 [a] (2)
| Entry | Additive | Yield (%) | D/L (%) |
| 1 | HOAt/DIC | 86.1 | 2.1 |
| 2 | HOBt/DIC | 78.8 | 8.9 |
| 3 | HOPO/DIC [b] | 88.5 | 45.1 |
| 4 | OxymaPure/DIC [b] | 89.8 | 3.8 |
[a] Couplings were conducted without preactivation, except in the case of entries 3 and 4. [b] A 2 min preactivation time was used.
Solid-Phase Peptide Synthesis (SPPS)
Racemization
Yield and racemization during the incorporation of Fmoc-Cys(Trt)-OH in solid-phase to render H-Gly-Cys-Phe-NH2 in DMF
| Entry | Additive | D/L (%) |
| 1 | HOAt/DIC | 0.1 |
| 2 | HOBt/DIC | 0.2 |
| 3 | OxymaPure/DIC | 0.1 |
Racemization during the incorporation of Fmoc-His(Trt)-OH in solid-phase to render H-Gly-His-Phe-NH2 in DMF (3)
| Entry | Coupling Reagent | D/L (%) |
| 1 | HOAt/DIC | 1.9 |
| 2 | HOBt/DIC | 5.1 |
| 3 | OxymaPure/DIC | 3.0 |
| 4 | Oxyma-B/DIC | 1.0 |
Synthesis of challenging/difficult peptides
Percentage purity of the pentapeptide H-Tyr-Aib-Aib-Phe-Leu-NH2 versus the tetrapeptide side-product des-Aib (H-Tyr-Aib-Phe-Leu-NH2 during solid-phase assembly (4)
| Entry | Coupling reagents | Pentapeptide (%) | des-Aib (%) [a] |
| 1 | DIC/HOBt | 8.4 | 83.1 |
| 2 | DIC/HOAt | 37.5 | 60.2 |
| 3 | DIC/OxymaPure | 42.8 | 50.4 |
| 4 | DIC/K-Oxyma | 71.0 | 6.7 |
| 5 | DIC/Oxyma-B | 26.4 | 61.1 |
[a]Deletion tetrapeptide (des-Aib) was identified by peak overlap in HPLC with an authentic sample obtained in solid phase.
Percentage of H-Tyr-MeGly-MeGly-Phe-Leu-NH2, and related deletion peptides, solid-phase assembly (2)
| Entry | Coupling Reagent | Pentapeptide (%) | des-MeGly (%) | des-Tyr (%) | H-MeGly-Phe-Leu-NH2 (%) |
| 1 | HOAt/DIC | 94.9 | 1.4 | 3.2 | 0.5 |
| 2 | HOBt/DIC | 84.8 | 7.5 | 6.6 | 1.1 |
| 3 | Oxyma/DIC | 91.4 | 3.8 | 4.2 | 0.6 |
Minimizing Side-Reactions
Aspartiimides can lead to both α and β peptides and their corresponding piperidides are difficult to detect. The formation of aspartiimides, which can take place during basic treatments (e.g. during piperidine treatment in a Fmoc/tBu strategy) can be reduced by adding OxymaPure®.
Reducing Side-Reactions: Aspartiimide Formation during the H-Ala-Orn-Asp-Gly-Tyr-Ile-NH2 synthesis (5)
| Concentration additive 20% piperidine/DMF | Additive | α-Peptide | β-Peptide | Aspartimide | Piperidides |
| 1M | HOBt | 79.00 | 0.25 | 14.03 | 6.72 |
| 1M | HOAt | 79.68 | 0.13 | 14.36 | 5.82 |
| 1M | OxymaPure® | 85.59 | 0.10 | 9.60 | 4.67 |
Addition of OxymaPure® to a piperidine solution during the Fmoc removal minimizes side-reactions such as aspartimide formation and double hit due to the presence of Pro.
Terminated Peptides
The presence of acetic acid can terminate the peptide chain.
According to Luxembourg Bio-Technology proprietary synthetic process, acetic acid is not used during the Oxyma Pure ® production, therefore no termination occurs during the synthesis.
| SPPS | Solution | |
| Coupling reagent | DIC, TBEC | DIC, EDC-HCl, TBEC, DCC |
| Protection | Fmoc/tBu | Fmoc/tBu, Z/tBu, Boc/Bn |
| Solvent | DMF, NBP, NOP, NMP, Me-THF, NBP-EtOAc (1:4), DMSO/2-Me-THF (2:8), DMSO/EtOAc (1:9), γ-Valerolactone (GVL) | DMF, Polar clean® |
| Stoichiometry (a) | AA-OxymaPure-Carbodiimide 3:3:3 | Protected-AA-EDC·HCl-OxymaPure |
| Fmoc Deprotection | 20% piperidine in DMF | |
| Coupling time | 2-5 min (microwave, heating), 30 min-1h RT | |
| Linker | Wang, Rink Amide, Sieber Amide, Ramage Amide, SASRIN, 2-CTC | - |
| Resin | Polystyrene (PS), TentaGel, Polyethylenglycol (PEG)-PS, ChemMatrix®, Li-resin | - |
a In both SPPS and in solution, the coupling can be carried out using in-situ activation or pre-activation.
In the in-situ mode, the protected amino acid and the OxymaPure are first added to the amino peptide and then the carbodiimide is added.
In the pre-activation mode, the protected amino acid, the OxymaPure, and the carbodiimide are left to react for 2-5 min and then the mixture is added to the amino component.
When possible the in-situ activation renders better yields. For the incorporation of protected Cys is better to use preactivation to minimize racemization.
Combination of OxymaPure® with other coupling reagents
To improve the efficiency of HBTU and PyBOP, it is recommended to add OxymaPure® to the reaction. In this case, the active specie will be the OxymaPure ester which is more reactive than the OBt ester.
OxymaPure® Compatibility with automated synthesis
OxymaPure® is compatible for use in automated synthesizers including microwave-assisted synthesis. OxymaPure®/DIC combination is stable at 900C of microwave conditions.